Structural snapshots of human pre-60S ribosomal particles before and after nuclear export

Xiaomeng Liang,Xiaomeng Liang,Xiaomeng Liang,Chengying Ma,Mei-Qing Zuo,Mei-Qing Zuo,Mei-Qing Zuo,Ningning Li,Ningning Li,Ning Gao,Ning Gao,Meng-Qiu Dong,Meng-Qiu Dong,Yunyang Zhang,Yunyang Zhang

doi:10.1038/s41467-020-17237-x

Abstract

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.

Highlights

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes
Our understanding of the general pictures of the assembly process and the diverse functions of assembly factors has been greatly advanced by cryo-electron microscopy structures of fungi pre-ribosomal particles at different assembly stages [reviewed in ref. 2,4]
Ribosome biogenesis is considered generally conserved in eukaryotes, it is currently unclear to what extent or which step of ribosome biogenesis differs among species

Summary

Results and discussion

Structures of pre-60S particles purified through NMD3. To obtain native pre-60S particles from human cells, we first generated a HEK293 cell line stably expressing a pre-60S nuclear export factor NMD319,20 with an affinity tag at the C-terminus (NMD3-tev-3XFLAG). These factors, located in the equivalent positions as seen in the yeast pre-60S particles, interact with their partners in a highly conserved fashion Such an example is GTPBP4, which is an organization hub interacting with multiple distant ribosomal proteins, assembly factors and rRNA helices along its path from the GTPase domain to the long C-terminal extension (Fig. 1a,b). As the particles transit from state A to B, a near-mature state of H89 is reached (Supplementary Movie 3) upon the release of GTPBP4 and TMA16 (Fig. 4f) In this state, NMD3-NTD becomes ordered likely due to the interaction with LSG1 (Fig. 4c)[23,24], and in particular, the specific interaction between the β4-β5 loop and the PTC (H92 and H90) is established. A new finding is that a stretch of evolutionarily conserved basic residues (R410 to K420) immediately downstream the RNAbinding OB domain of NMD3, not sufficiently resolved for atomic modeling though, is clearly seen to be embedded in the

H89 State A H89 State B H89 State C

Methods

A A A 3901